Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen resistant to certain antibiotics that are otherwise effective against methicillin-sensitive S. aureus (MSSA). More specifically, strains of S. aureus that are oxacillin and methicillin resistant, historically termed MRSA, are resistant to all β-lactam agents, including cephalosporins and carbapenems. Hospital-associated MRSA isolates often are multiply resistant to other commonly used antimicrobial agents, including erythromycin, clindamycin, and tertacycline, while community-associated MRSA isolates are often resistant only to β-lactam agents and erythromycin. (See U.S. CDC publication, “Laboratory Detection of: Oxacillin/Methicillin-Resistant Staphylococcus aureus” (2005)) The estimated number of people developing a serious MRSA infection (i.e., invasive) in 2005 was about 94,360. (JAMA 298:1763-1771 (2007))
The magnitude of the MRSA problem is growing, and the human and economic tolls are rising. Indeed, approximately 32% of the U.S. population is already colonized with S. aureus, and approximately and 0.8% is already colonized MRSA. (Kuehnert et al., J. Infect. Diseases 193:172 (2006)) The proportion of healthcare-associated staphylococcal infections due to MRSA has also been increasing: 2% of S. aureus infections in U.S. intensive-care units were MRSA in 1974, 22% in 1995, and 64% in 2004. (Klevens et al., Clin. Infect. Diseases 42:389 (2006)) The evidence indicates that infections by MRSA and MSSA are associated with similar direct medical costs, but that MRSA infection is associated with more than double the rate of death when compared to infection by MSSA (i.e., 21% versus 8%). (Rubin et al., Emerg. Infect. Diseases 5:9 (1999)) Various diagnostic and screening assays that detect MRSA have been developed to support early intervention.
Although screening for MRSA colonization has traditionally relied on culture of specimens, for example using selective broth or agar medium, molecular approaches have been developed to speed the time to diagnosis. For example, Huletsky et al., in J. Clin. Microbiol. 42:1875 (2004) described a real-time PCR assay for detecting MRSA in specimens containing a mixture of staphylococci. The assay relied on detection of a mobile genetic element, designated the Staphylococcal Cassette Chromosome mec (SCCmec), integrated at the 3′ end of an open reading frame of unknown function, termed “orfX.” The SCCmec, which carries the mecA gene that confers drug-resistance in MRSA bacteria, is located at least 9 kb distant from the integration junction detected in the amplification reaction. The technique described by Huletsky et al., requires a collection of five primers that hybridize on one side of the SCCmec right extremity sequence, and one primer and three molecular beacon hybridization probes specific for the S. aureus chromosomal orfX gene. The requirement for numerous orfX primers reflects the known sequence divergence among different MRSA isolates. Alternative molecular assays independently detect a first gene sequence specific for S. aureus, and a second gene sequence specific for the mecA gene, but fail to establish physical linkage between the two sequences.
Published reports have highlighted certain deficiencies in each of the existing molecular approaches for MRSA detection. For example, Desjardins et al., in J. Clin, Microbiol. 44:1219 (2006) described results from procedures carried out using a commercial MRSA assay employing the junction-based amplification procedure for testing nasal specimens. Briefly, the authors noted the recovery of MSSA isolates from samples identified as being MRSA-positive in the real-time assay. This incidence of false-positive results might be explained by the presence of a junction in bacteria that have lost the mecA gene, for example as the result of genetic instability in the absence of antibiotic selective pressure. Moreover, our coworkers have observed that the same commercial assay failed to detect at least one highly drug resistant MRSA isolate, presumably because the junction sequences were highly diverged and did not hybridize amplification primers efficiently. The utility of the screening strategy is further confounded by the fact that the SCC junction is not uniquely associated with the methicillin resistance (see Becker et al., in J. Clin. Microbiol, 44:229 (2006), on page 231). Thus, the junction-based MRSA detection approaches are plagued by both false-positive and false-negative results.
Other molecular approaches for MRSA screening have been considered, but also suffer deficiencies. For example, Becker et al., in J. Clin. Microbiol. 44:229 (2006) questioned whether nasal colonization by both methicillin-resistant coagulase-negative staphylococci (MR-CoNS) and MSSA strains occur frequently enough to represent a risk of false-positive MRSA determinations by molecular methods. The authors showed that 3-4% of a sample population of patients admitted for hospital procedures showed evidence for nasal colonization by MSSA and MR-CoNS. Thus, samples from colonized individuals included both mecA DNA sequences, and S. aureus-specific DNA sequences. The study concluded that a molecular MRSA screening test independently targeting the mecA gene and a S. aureus-specific gene would be associated with an unacceptable positive predictive value of 40% when applied directly to clinical specimens in a low MRSA setting.
Existing approaches for MRSA screening ofcomplex samples, such as nasal swab samples, are prone to certain levels of erroneous assignments. Accordingly there remains a need for a rapid molecular approach that reduces false-positive MRSA assignments, particularly in instances of coinfection by S. aureus and methicillin-resistant bacteria other than S. aureus (e.g., MR-CoNS). The present invention answers this need.